Quantification and Comparison of Signals Generated by Different FRET-Based cAMP Reporters
A variety of FRET-based biosensors are currently in use for real-time monitoring of dynamic changes of intracellular cAMP. Due to differences in sensor properties, unique features of the cell type under examination and diverse specifications of the imaging setups in different laboratories, data generated using these sensors may not be immediately comparable within the same study or across studies. To facilitate comparison, often FRET data are normalized and expressed as fractional change of the maximal FRET response at sensor saturation. However, this approach may lead to misinterpretation of the underlying cAMP change. In this chapter, we provide examples of the problems that may arise when using normalized FRET data and present a method based on the conversion of FRET ratio changes into actual cAMP concentrations that mitigates these issues.
Key wordsFluorescence resonance energy transfer FRET Biosensors cAMP Protein kinase A Real time imaging Intracellular signaling
This work was supported by the British Heart Foundation (PG/10/75/28537 and RG/17/6/32944) and the BHF Centre of Research Excellence, Oxford (RE/13/1/30181).
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